The unmethylated CpG motifs present in bacterial DNA interact with toll-like receptor 9 to trigger a pro-inflammatory immune response. CpG DNA also improves antigen presenting cell function, thereby facilitating the development of adaptive immunity. My laboratory established that synthetic oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN) could be conjugated to apoptotic tumor cells to generate tumor vaccines that were rapidly internalized by professional APCs, promoted DC maturation, and boosted the induction of tumor-specific immunity. In multiple murine models we found that vaccination with CpG-conjugated apoptotic cell vaccines significantly reduced susceptibility to tumor challenge. This effect was observed both in mice vaccinated and then challenged and in animals immunized up to 5 weeks post challenge. The addition of agents known to boost NK and T cell activation, such as 4-1BB MAb, synergistically enhanced the anti-tumor effect of CpG ODN. The general utility of this approach was established by testing 6 different tumor types and showing activity in each case. Unfortunately, the ability of these vaccines to eradicate tumors waned as tumor burden increased. We believe this reflects the ability of large established tumors to generate an immunosuppressive microenvironment capable of inhibiting Ag-specific cellular responses that interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near established cancers and have been shown to inhibit the activity of antigen-specific T and NK cells. Our studies demonstrate that when CpG ODN are injected into the tumor, they immunosuppressive activity of monocytic (CD11b+, Ly6G neg, Ly6C high) MDSC is significantly reduced. We hypothesize that the signal provided via TLR9 ligation is sufficient to overcome the inhibitory signals provided by the tumors cells. Monocytic MDSC express TLR9 and respond to CpG stimulation by i) losing their ability to suppress T cell function, ii) producing Th1 cytokines and iii) differentiating into M-1 like macrophages with tumoricidal capability. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and support intra-tumoral injection as the optimal route for their delivery. We have extended these studies to include mMDSC from normal human donors and cancer patients. Results show that stimulation with the appropriate TLR agonist induces human mMDSC to mature and lose their immunosuppressive activity. Of interest, CpG ODN are relatively inactive on human MDSC whereas TLR 7/8 agonists reproduce on human MDSC the activity that TLR9 ligation has on mouse MDSC. Thus, we conclude that a combination of TLR ligands can be being harnessed to achieve two independent but mutually supportive functions: boosting the efficacy of anti-tumor vaccines and reducing the activity of cells at the tumor site that would otherwise reduce the efficacy of this anti-tumor response. Our ongoing research aims to identify the optimal therapeutic window for the delivery of CpG ODN and other TLR ligands and examine whether the protective immune responses they elicit can be accelerated and/or magnified by combining them with other immunomodulatory agents (such as additional TLR ligands and small molecule agonistic immune potentiators). Efforts to optimize the therapeutic utility of CpG ODN require a detailed understanding of the cells they activate (both directly and indirectly), their duration of action, and the regulatory pathways involved in mediating these responses. To clarify these issues, we are using microarray technology to identify the genes and networks central to the immune stimulation elicited by CpG ODN. Such experiments are conducted in vitro on highly purified cell subpopulations (including human pDC and MDSC) and in vivo studies of mice to monitor gene expression under physiologic conditions. Recent results show that a subset of genes characterized by shared anti-viral activity was consistently up-regulated by ODNs that otherwise mediate discrete functions. This group of genes was largely dependent on autocrine type I interferon (IFN) signaling, as their induction was blocked by neutralizing antibody targeting the type I IFN receptor. Coupling these experiments with a meta-analysis of other published works led to the identification of a set of 32 functionally conserved genes that was reproducibly activated by different types of CpG DNA in different species and cell types. Functionally, these core genes support a type I IFN response to viral infection, and differ from genes up-regulated by only a single type of CpG ODN. These findings help define the conserved and sequence-specific patterns of gene activation triggered via TLR9 and improve our understanding of the immunomodulatory effects elicited by CpG ODN. Analysis of MDSC isolated from normal human donors and patients with various types of cancer demonstrate that exposure to TLR 7/8 agonists uniformly induce such cells to mature into tumoricidal macrophages without suppressive activity. These findings increase the likelihood that combination TLR agonist therapy will find utility in humans.